Holistic monitoring of Campylobacter jejuni biofilms with NanoLuc bioluminescence

Abstract

Campylobacter jejuni, a major cause of foodborne zoonotic infections worldwide, shows a paradoxical ability to survive despite its susceptibility to environmental and food-processing stressors. This resilience is likely due to the bacterium entering a viable but non-culturable state, often within biofilms, or even initiating biofilm formation as a survival strategy. This study presents an innovative application of NanoLuc bioluminescence to accurately monitor the development of C. jejuni biofilms on various substrates, such as polystyrene plates, mucin-coated surfaces, and chicken juice matrices. Introduction of NanoLuc luciferase in a pathogenic C. jejuni strain enables rapid non-invasive holistic observation, capturing a spectrum of cell states that may comprise live, damaged, and viable but non-culturable populations. Our comparative analysis with established biofilm quantification methods highlights the specificity, sensitivity, and simplicity of the NanoLuc assay. The assay is efficient and offers precise cell quantification and thus represents an important complementary or alternative method to conventional biofilm monitoring methods. The findings of this study highlight the need for a versatile approach and suggest combining the NanoLuc assay with other methods to gain comprehensive insight into biofilm dynamics. KEY POINTS: • Innovative NanoLuc bioluminescence assay for sophisticated biofilm quantification. • Holistic monitoring of C. jejuni biofilm by capturing live, damaged and VBNC cells. • Potential for improving understanding of biofilm development and structure.

Keywords: Campylobacter jejuni; Biofilm quantification; Food safety; Foodborne infections; NanoLuc bioluminescence; VBNC.

Fig. 1

Bioluminescence intensity represented as relative light units (RLU) as a function of concentration (CFU/mL) of C. jejuni expressing NanoLuc (i.e., containing the plasmid pMW10_nLuc; red) or control C. jejuni (i.e., containing the empty plasmid pMW10; black)

Fig. 2

Monitoring biofilm formation on polystyrene plates over 72 h using the NanoLuc bioluminescence assay (A), plate counting assay (B), crystal violet staining (C), and resazurin fluorescence (D). Initial inocula concentrations of 2.50 × 10⁷, 6.00 × 10⁷, 1.00× 10⁸, and 3.00 × 10⁸ CFU/mL are denoted as K1, K2, K3, and K4, respectively. Each cultivation was performed in three biological and three technical replicates. Error bars denote standard deviation, and the horizontal dashed black lines denote the limit of detection (NanoLuc) or minimal reliable signal (crystal violet and resazurin). logC, logarithm of CFU/mL; FI, fluorescence intensity; A₅₈₄, absorbance at 584 nm

Fig. 3

Monitoring biofilm formation on mucin-coated polystyrene plates over 72 h using the NanoLuc bioluminescence assay (A) and plate counting assay (B). Mucin was applied at concentrations of 1 mg/mL (M1), 500 µg/mL (M2), and 50 µg/mL (M3). Each cultivation was performed in three biological and three technical replicates. Error bars denote standard deviation, and the horizontal dashed black lines denote the limit of detection (NanoLuc). logC, logarithm of CFU/mL

Fig. 4

Monitoring biofilm formation in chicken juice over 48 h using the NanoLuc bioluminescence assay (A) and plate counting assay (B). Mueller–Hinton broth was used as a control. Error bars represent standard deviation of six biological replicates in three technical replicates (chicken juice) or three biological replicates in three technical replicates (controls). The horizontal dashed black line denotes the limit of detection (NanoLuc). logC, logarithm of CFU/mL

Fig. 5

Monitoring Campylobacter jejuni quantity during biofilm formation in a mixed culture with Salmonella enterica over 48 h using the NanoLuc bioluminescence assay (A) and the plate counting assay (B). Error bars represent standard error of six biological replicates and three technical replicates, and the horizontal dashed black lines denote the limit of detection (NanoLuc). logC, logarithm of CFU/mL